The enhanced entry of extracellular Ca2+ is a major component of cellular Ca2+ signals generated by a variety of hormones and neurotransmitters acting on receptors coupled to phospholipase C (PLC). The focus of our research is to understand the nature of this Ca2+ entry, and its roles in overall intracellular signaling mechanisms. Interest in this field has been largely dominated by so-called store- operated Ca2+ channels (e.g. the CRAC channels) whose gating is entirely dependent on, and subsequent to, the depletion of intracellular Ca2+ stores. More recently however, other store- independent pathways have been shown to play a key role - particularly at lower, more physiologically relevant, levels of stimulation. Of these, the arachidonic acid-regulated Ca2+-selective (ARC) channels, that we first described some 8 years ago, remain the most thoroughly characterized. A major impediment to study of both the CRAC channels and the ARC channels has been the lack of any information regarding the molecular nature of these channels. In the past 2-3 years, this situation has been fundamentally transformed by the discovery of the STIM and Orai proteins. Thus, it has been shown that STIM1 located in the membrane of the endoplasmic reticulum, senses the depletion of intracellular Ca2+ stores, and activates the CRAC channels whose pore is comprised of a homotetramer of Orai1 subunits. Surprisingly, we have recently shown that STIM and Orai proteins also function in parallel, yet entirely distinct, ways to affect ARC channel activity. ARC channel activity is regulated by STIM1, but it is the pool of this protein that is resident in the plasma membrane that is responsible, and the ARC channel pore is comprised of a heteromeric complex of both Orai1 and Orai3 subunits. These close molecular relationships indicate that the store-operated CRAC channels and the store-independent ARC channels represent the founding members of an entirely new family of channels - the "Orai-based channels". However, our functional studies have demonstrated that these two channels evolved to operate under distinct conditions of stimulation and to serve unique roles in the regulation of agonist-activated Ca2+ signals. Importantly, these new molecular insights have created a wealth of novel tools and approaches that we now propose to use to determine the detailed molecular organization of the ARC channel pore (Aim 1), the molecular mechanisms underlying activation of the channels by arachidonic acid (Aim 2), the molecular basis for their regulation by PKA-dependent phosphorylation (Aim 3), and the mechanisms by which their activity acts to modulate the oscillatory Ca2+ signals generated in cells stimulated with low agonist concentrations (Aim 4). PUBLIC HEALTH RELEVANCE: The receptor-activated entry of calcium ions into cells represents a key component in the generation of intracellular calcium signals that are known to regulate a host of different cellular functions - including cell division and proliferation, secretion, motility, and cell death. Misregulation, or errors in these signals underlie a variety of different disease states including cancer, immunodeficiency, pancreatitis, and various muscular and neuronal diseases. Defining the molecular bases of the relevant entry pathways and their regulation, will undoubtedly help elucidate the critical mechanisms involved in their activity, and identify potential targets for their clinical manipulation.